A major problem attendant to studies of central nervous system tissue is the maintenance of cell viability of such tissues. The inability to maintain central nervous system tissue viability in culture for prolonged periods of time and under various environmental conditions has impeded the development of effective therapeutic regimens for treating central nervous system disorders.
A nutrient balanced salt solution (medium) for maintaining central nervous system tissue viability in a high-carbon dioxide atmosphere (5% CO.sub.2) has recently been developed. That medium, Neurobasal.TM. (Gibco/Life Technologies, Inc., Gaithersburg, Md.), is a bicarbonate buffered medium optimized for the growth of embryonic rat hippocampal neurons at a pH of 7.3 in 5% CO.sub.2. Neurobasal.TM. is a derivative of Dulbecco's Modified Eagle's Medium (DMEM) and was formulated to optimize embryonic rat hippocampal cell survival. When compared to DMEM, Neurobasal.TM. has less NaCl and less NaHCO.sub.3, resulting in a lower osmolality, and lesser amounts of cysteine and glutamine, resulting in diminished glial growth. In addition, Neurobasal.TM. contains alanine, asparagine, proline and vitamin B12, all of which are absent from DMEM.
Although neurons can be maintained in a 5% CO.sub.2 atmosphere in this high bicarbonate medium, when supplemented with B27 (a hormone and anti-oxidant supplement available from Life Technologies, Inc.), neurons undergo rapid death when transferred to ambient CO.sub.2 (0.2%) conditions. Death is associated with a rapid rise in medium pH to a value of 8.1.
The preparation and study of neural tissue and cells frequently requires the use of ambient CO.sub.2 levels outside of an incubator. Existing methods for controlling the pH of cells outside of incubators include the use of weak buffers (e.g., as found in Dulbecco's modified Eagle's medium or L 15 medium) and the use of continuous gassing with 5-10% CO.sub.2 to maintain physiological pH.
A simple test, however, shows that ambient CO.sub.2 causes the pH of DMEM to quickly rise to a value of 8.1 outside the incubator. The common practice of buffering with HEPES slows but does not prevent this substantial alkalinization. The practice of continuously gassing tissues to maintain high CO.sub.2 levels and physiological pH is cumbersome and expensive. There continues to be a need in the art, therefore, for a medium that can maintain physiological pH and neural cell viability in ambient CO.sub.2 conditions.